Vascular occlusions caused by blood clots such as thrombi and embolisms are serious medical maladies that can become limb or life threatening if not timely treated. Devices and methods have been developed for the treatment and removal of vascular blood clots. By way of illustration, see U.S. Pat. No. 4,447,236 (Quinn), issued May 8, 1984; U.S. Pat. No. 4,692,139 (Stiles), issued Sep. 8, 1987; U.S. Pat. No. 4,755,167 (Thistle et al.), issued Jul. 5, 1988; U.S. Pat. No. 5,167,628 (Boyles), issued Dec. 1, 1992; U.S. Pat. No. 5,222,941 (Don Michael), issued Jun. 29, 1993; U.S. Pat. No. 5,250,034 (Appling et al.), issued Oct. 5, 1993: U.S. Pat. No. 5,370,653 (Cragg), issued Dec. 6, 1994; U.S. Pat. No. 5,380,273 (Dubrul et al.), issued Jan. 10, 1995; U.S. Pat. No. 5,498,236 (Dubrul et al.), issued Mar. 12, 1996; U.S. Pat. No. 5,626,564 (Zhan et al.), issued May 6, 1997; U.S. Pat. No. 5,709,676 (Alt), issued Jan. 20, 1998; U.S. Pat. No. 5,865,178 (Yock), issued Feb. 2, 1999, and WO 90/07352 (published Jul. 12, 1990). Such methods and devices include infusion catheters for delivering thrombolytic or fibrinolytic agents to the blood clot and dissolving it. Infusion catheters are typically used in conjunction with enzymatically active agents that are capable of degrading the fibrin in the clot and thus effectively dissolving the clot. Such enzymes are typically referred to as “thrombolytic” or “fibrinolytic” agents.
Fibrolase is a known fibrinolytic zinc metalloproteinase that was first isolated from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix). See Guan et al., Archives of Biochemistry and Biophysics, Volume 289, Number 2, pages 197–207 (1991); Randolph et al., Protein Science, Cambridge University Press (1992), pages 590–600; European Patent Application No. 0 323 722 (Valenzuela et al.), published Jul. 12, 1989; and U.S. Pat. No. 4,610,879 (Markland et al.), issued Sept. 9, 1986. Fibrolase has been shown to be fibrinolytic, and this metalloproteinase has been documented to have proteolytic activity against the fibrinogen Aα-chain, with reduced proteolytic cleavage of the Bβ-chain and no activity against the γ-chain of fibrinogen; Ahmed et al., Haemostasis, Volume 20, pages 147–154 (1990). Because fibrin is a principal component of blood clots, the fibrinolytic properties of fibrolase point to its potential as a clot dissolving agent for in vivo thrombolytic use; see Markland et al., Circulation, Volume 9, Number 5, pages 2448–2456 (1994), and Ahmed et al., above.
Novel Acting Thrombolytic (NAT) is a modified form of fibrolase that differs from fibrolase in that NAT contains 201 amino acids with an N-terminal sequence of SFPQR, whereas the N-terminal sequence of native fibrolase begins with EQRFPQR and is 203 amino acids in length. The amino-terminal modification was designed to prevent chemical reactions at amino acid residues that were capable of forming a variable quantity of cyclized glutamine (pyroglutamic acid) which have the potential to create lot-to-lot variations in quality and uniformity of the product. Thus, NAT can be viewed as a more stable molecule.
Despite these structural differences, NAT and fibrolase are similar with respect to enzymatic (fibrinolytic) activity. This similarity in biological activity is consistent with data indicating that the active site of the fibrolase molecule spans amino acids 139–159, as described by Manning in Toxicon, Volume 33, pages 1189–1200 (1995), and its predicted location in three-dimensional space is distant from the amino-terminus. The active site of the fibrolase and NAT molecules contains a zinc atom which is complexed by three histidine residues.
Published literature on venom-derived fibrolase has demonstrated its proteolytic activity against fibrinogen at the Lys413-Leu414 site and against the oxidized β-chain of insulin at the Ala14-Leu15 site; Retzios and Markland, Thrombosis Research, Volume 74, pages 355–367 (1994); Pretzer et al., Pharmaceutical Research, Volume 8, pages 1103–1112 (1991), and Pretzer et al., Pharmaceutical Research, Volume 9, pages 870–877 (1992). NAT has also been determined to have proteolytic activity on these substrates at the same cleavage sites.
In contrast to fibrinolytic metalloproteinases such as fibrolase and NAT, clot lysing agents such as streptokinase, urokinase and tissue-type plasminogen activator (tPA) are plasminogen activators which promote thrombolysis by activation of the endogenous fibrinolytic system. More specifically, plasminogen activators catalyze the conversion of plasminogen into plasmin, a serine protease. Plasmin is capable of cleaving fibrinogen and fibrin at arginyl-lysyl bonds, and it is through the generation of plasmin that the plasminogen activators ultimately affect fibrin degradation and clot lysis. Current commercially available thrombolytic agents are plasminogen activators, such as urokinase, streptokinase or tPA.
Unlike the plasminogen activator class of thrombolytic drugs, fibrinolytic metalloproteinases, such as fibrolase and NAT, do not rely on the endogenous fibrinolytic system (conversion of plasminogen to plasmin). Hence, this class of clot lysing agents can be distinguished from the plasminogen activators by their unique mode of action and are defined as “direct” fibrinolytic agents.
Alpha2-macroglobulin is a prevalent proteinase inhibitor present in mammalian serum and is one of the largest of the serum proteins (having a molecular weight of 725 kilodaltons). The specificity of α2-macroglobulin for proteinases is broad, encompassing serine, cysteine, aspartic and metalloproteinase classes. The α2-macroglobulin molecule is a tetramer of identical subunits that are disulfide bonded in pairs with a non-covalent association of the half molecules. Thus, under reducing conditions, native α2-macroglobulin can be dissociated into its four monomeric subunits.
Each subunit of α2-macroglobulin possesses a region that is very susceptible to proteolytic cleavage (the “bait” region). Proteolysis of the bait region induces a conformational change in α2-macroglobulin, which entraps the proteinase within the α2-macroglobulin molecular structure. This process is described in the literature as a “venus fly-trap” interaction. Once the proteinase is entrapped, it is sterically hindered and therefore cannot access its macromolecular substrate.
In addition, a covalent bond can form between α2-macroglobulin and many of the proteinases that it entraps. As mentioned, entrapment of a proteinase induces a conformational change in the α2-macroglobulin molecule. It is presumed that upon this conformational change, a thioester bond on the interior of the α2-macroglobulin molecule becomes reactive and can form a covalent bond with nucleophilic residues (such as lysine) of the entrapped proteinase. Thus, within the general circulation, α2-macroglobulin can effectively neutralize a variety of proteinases.
Moreover, the conformational change in α2-macroglobulin brought about by the entrapment of a proteinase results in a form that is recognized by the reticuloendothelial system. Clearance of α2-macroglobulin-entrapped proteinases is generally described with half-life values in minutes and is believed to occur through the low-density lipoprotein (LDL)-receptor related protein expressed on macrophages, hepatocytes and fibroblasts. For more on α2-macroglobulin, see Methods in Enzymology, edited by A. J. Barrett, Academic Press, Inc., Philadelphia, (1981), pages 737–754.
Alpha2-macroglobulin is capable of forming a macromolecular complex with fibrolase, NAT and other proteinases. Unlike some proteinases that can form a dissociable complex with α2-macroglobulin, fibrolase and NAT are two examples of fibrinolytic metalloproteinases that form a complex which cannot be dissociated from α2-macroglobulin under physiologic conditions. When purified human α2-macroglobulin and NAT, for instance, are incubated together, formation of the complex begins in seconds and is nearly complete within a few minutes. This phenomenon shows that in vitro complex formation can be rapid and is suggestive of the potential rapidity of complex formation between α2-macroglobulin and NAT or other fibrinolytic metalloproteinases in vivo.
Although α2-macroglobulin is one of the major plasma proteins, there is nonetheless a finite quantity of α2-macroglobulin in the circulation that would be available to bind and neutralize a fibrinolytic metalloproteinase. The α2-macroglobulin binding capacity is therefore saturable. Once the α2-macroglobulin binding capacity has been exceeded, the concentration of unbound fibrinolytic metalloproteinase rises proportionally as additional fibrinolytic metalloproteinase is added to the sample.
The presence of α2-macroglobulin in the general circulation of a patient presents a challenge for the systemic (for example, intravenous) administration of fibrolase, NAT and other fibrinolytic metalloproteinases that are bound up by α2-macroglobulin in the general blood circulation. Unless the saturable level of innate α2-macroglobulin is exceeded by the systemically administered dose of such fibrinolytic metalloproteinases, the latter will effectively be neutralized and rendered ineffective for therapeutic purposes.
In one in vivo study, conducted in rabbits, the biological effectiveness of venom-derived fibrolase was examined following systemic intravenous administration. Ahmed et al., Haemostasis, above. The dose of fibrolase used was 3.7 milligrams per kilogram, which was estimated to yield a final blood concentration of approximately 60 micrograms per milliliter in a 3.0-kilogram rabbit. This amount was chosen based on studies examining the inactivation of the enzyme in the presence of blood or plasma, presumably due to α2-macroglobulin (see pages 336 and 339).
In another in vivo study, the biological effect of recombinant fibrolase on clot lysis was examined in canines. Markland et al., Circulation, above. Four milligrams of this material per kilogram (of animal body weight) was infused over five minutes proximal to a pre-induced thrombus in the left carotid artery via a catheter device (see page 2450). Here again, it was noted that inactivation of fibrolase occurs in the general blood circulation presumably due to the presence of α2-macroglobulin (see page 2454, second column, last paragraph).
As these two studies show, the deactivating effects of α2-macroglobulin can be overcome by either administration or systemic dosages of fibrinolytic metalloproteinase that exceed the saturable level of innate α2-macroglobulin (the rabbit study) or by delivering the enzyme locally to the site of the clot (the dog study) and avoiding systemic administration. On the other hand, doses of the fibrinolytic metalloproteinase in excess of the saturable level of α2-macroglobulin, whether delivered systemically or locally, may exceed levels that are safe and well tolerated by the subject being treated. Notably, fibrinolytic metalloproteinases are capable of destroying not only fibrin, but they may also degrade other structural proteins and are therefore potentially toxic in vivo when present in large amounts that exceed the saturable level of α2-macroglobulin.
The formation of a blood clot or other fibrin-based occlusion is also a concern when using an indwelling catheter or other vascular access device. There are many conditions that require recurrent or prolonged use of a vascular access device, such as a catheter, access graft, sheath, needle, arteriovenous fistula or shunt. For example, various procedures associated with hemodialysis, chemotherapy, blood infusion or exchange and other procedures involving recurrent intravenous or intraartieral drug delivery (or fluid withdrawal) may involve the use of indwelling catheter or other permanent or semi-permanent implanted medical device. Blood clots may form in or around, or attach to, any device that has been introduced into a vascular space, particularly where the device remains in the vascular space for an extended period of time or where the device has one or more very small openings. In addition, other fibrin-based occlusions may attach to any portion of an indwelling vascular access device which could prevent or hinder the proper functioning of the device.
It would be useful to have a fast and efficient method to treat, i.e., destroy, dissolve or lyse clots or other occlusions that have formed in, around or attached to, an indwelling vascular access device. In the absence of an efficient method to treat clots that have formed in or around a vascular access device, the indwelling device may have to be replaced by a physician, incurring additional cost and risk to the patient.
Accordingly, it is an object of the present invention to provide a safe and effective method for treatment of a blood clot or occlusion in or around an indwelling vascular access device.
It is also an object of the present invention to provide a method for restoring patency to a fully or partially occluded indwelling vascular access device.
It is a further object of the present invention to provide a method for restoring function to an indwelling vascular access device where function has been altered by a fibrin-based occlusion.
It is also an object of the present invention to provide a safe and biologically effective way of using locally administered fibrinolytic metalloproteinases to lyse blood clots in vivo.